免疫不育疫苗的研究进展

免疫不育疫苗主要以哺乳动物的精子或卵子蛋白以及在受精和胚胎早期发育过程中发挥重要作用的激素为靶抗原。以激素为抗原的不育疫苗产生的不育效果多为不可逆的,且对机体损伤较大。以精子表面抗原制备的疫苗能够诱导产生精子抗体和不育效果,目前已成为避孕研究的一个热点。哺乳动物卵透明带（zona pellucida,ZP）是覆盖于卵母细胞及着床前受精卵外的一层基质,其在调节精卵特异性结合、诱导获能精子发生顶体反应和阻止多精受精等方面发挥着重要作用。ZP相对分子质量较小且免疫原性强,是免疫不育疫苗理想的靶抗原,抗ZP抗体可阻断精卵结合,故可被用作人类避孕和免疫不育控制有害动物种群数量的靶抗原,但人用ZP疫苗免疫机体后造成的卵巢功能损伤和免疫抑制等问题尚有待明确。     

Economic Benefit of Fertility Control in Wild Horse Populations

ABSTRACT I projected costs for several contraceptive treatments that could be used by the Bureau of Land Management (BLM) to manage 4 wild horse (Equus caballus) populations. Potential management alternatives included existing roundup and selective removal methods combined with contraceptives of different duration and effectiveness. I projected costs for a 20-year economic life using the WinEquus© wild horse population model and state-by-state cost estimates reflecting BLM's operational expenses. Findings revealed that 1) currently available 2-year contraceptives in most situations are capable of reducing variable operating costs by 15%, 2) experimental 3-year contraceptives may be capable of reducing costs by 18%, and 3) combining contraceptives with modest changes to herd sex ratio (e.g., 55–60% M) could trim costs by 30%. Predicted savings can increase when contraception is applied in conjunction with a removal policy that targets horses aged 0–4 years instead of 0–5 years. However, reductions in herd size result in greater variation in annual operating expenses. Because the horse program's variable operating costs make up about half of the total program costs (which include other fixed costs), contraceptive application and management can only reduce total costs by 14%, saving about $6.1 million per year. None of the contraceptive options I examined eliminated the need for long-term holding facilities over the 20-year period simulated, but the number of horses held may be reduced by about 17% with contraceptive treatment. Cost estimates were most sensitive to the oldest age adoptable and per-day holding costs. The BLM will experience significant cost savings as carefully designed contraceptive programs become widespread in the wild horse herds it manages.     

Ultrastructural studies of the porcine zona pellucida during the solubilization process by Li-3,5-diiodosalicylate

Isolated porcine zonae pellucidae were investigated using transmission electron microscopy. It was found that the fine structure of the zona is not homogenous. The region near the oocyte consists of a more tightly packed micellar structure than the structure near the external surface. However, no clear boundary between the two structural features could be detected. The assumption that the molecular structure of the external and internal surface of the zona is not the same is substantiated by the finding that antibodies against the whole zona structure react apparently only with the more loosely bound structure on the external surface. This effect is attributed to differences in the spacial arrangements of the micellar structures rather than to differences in chemical composition. Solubilization of the zona results in a reorientation of the otherwise randomly interconnected structure. At lower Li-3,5-diiodosalicylate concentration (0.05 M) the fibrils seems to expand so that the individual fibers lie almost parallel to each other. At a higher Li-3,5-diiodosalicylate concentration (0.2 M) the individual micelles begin to break up from the zona surface, while at a still higher concentration (0.3 M) the rigid structure of the zona is completely solubilized. In the latter case no residual material could be detected in the sediment following high speed centrifugation of the mixture. These electron microscopic results correlated with the protein concentrations in the supernatant indicating that the maximal protein content (35 ng/zona) is obtained at 0.3 M or higher Li-3,5-diiodosalicylate concentrations.     

Mapping of B cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (Trichosurus vulpecula)

Immunocontraceptive vaccines against zona pellucida (ZP) proteins are being developed for brushtail possum (Trichosurus vulpecula) management in New Zealand. Mapping of B cell epitopes on the ZP2 protein of possums was undertaken in this study to define the antigenic regions that may be crucial to sperm-egg binding. The amino acid sequence of the full-length possum ZP2 protein (712 amino acids) was used to synthesize a complete set of 71 (15-mer) biotinylated peptides with an offset of five amino acids. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with recombinant possum ZP2 (rZP2) constructs. Seventeen continuous epitopes were located on possum ZP2 protein. Comparisons of the peptide binding pattern of antibodies in individual sera with the fertility status of the same immunized possums revealed three significant infertility-relevant peptide epitopes (amino acids 111-125, 301-315, and 431-445). One of these (amino acids 431-445) bound to possum spermatozoa from the caudal epididymis. The implications of these findings for developing immunocontraceptive vaccines for possum control are discussed.     

Interaction of boar spermatozoa with porcine oocytes: Increase in proteins with high affinity for the zona pellucida during epididymal transit

Methods for the investigation of cell-associated calcium and intracellular calcium were studied in washed ejaculated human spermatozoa. Experiments using ⁴⁵Ca²⁺ indicated that human spermatozoa were permeant to calcium and that a significant proportion of the cellassociated calcium (approximately 50%) was accumulated in the mitochondrion. This necessitated the use of alternative procedures to measure cytoplasmic free calcium. The ability of human spermatozoa to accumulate and de-esterify the intracellular fluorescent calcium indicator Quin-2 was established. Using this technique, the resting level of free intracellular calcium in human spermatozoa was found to be 146.0 ± 19.9 nM, and was significantly elevated upon addition of the divalent cation ionophore ionomycin. In further experiments designed to illustrate the applications of the Quin technique, data was obtained suggesting that the mechanisms controlling intracellular calcium in human spermatozoa are temperature dependent but do not involve voltage-sensitive calcium channels.     

Ovoperoxidase activity in ionophore treated mouse eggs. II. Evidence for the enzyme's role in hardening the zona pellucida

One consequence of fertilization or parthenogenetic activation of mammalian eggs is an altaration in the solubility proprieties of the zona pellucida, known as zona hardening. Several lines of evidence indicate that an ovoperoxidase, which is activated and/or secreted from mouse eggs. Following parthenogenetic activation, corss-links tyrosine residues in the zona pellucida and results in hardening of the zona. First, zona hardening, as determined by decreased solubility of the zona in pronase, is inhibited by several compounds known to inhibit peroxidases. Inhibitors of hardening include phenylhydrazine, sodium sulfite, sodium azide, and glycine ethyl ester. Second, tyrosine analogs inhibit zona hardening, unless the phenolic hydroxyl group or ortho position is blocked. That is, O-methyltyrosine (methyl substitution of phenolic hydroxyl) does not inhibit hardening; o-methyltyrosine (methyl substitution of one ortho position) partially inhibits, whereas tyramine and N-acetyltyrosine (free hydroxyl and ortho positions) effectively block hardening. Finally, exogenous horseradish peroxidasepromotes limited hardening of the zona in unactivated eggs. These results are consistent with a peroxidase catalyzed cross-linking of tyrosines in the zona that results in hardening of the zona pellucida.     

Comparative cytochemistry of the developing ovarian follicles of the dog,rabbit, and mouse: Origin of the zona pellucida

The reactivity of the glycoproteins of ovarian follicles of the dog, rabbit, and mouse were compared, using the Alcian blue (pH 0.5) and Alcian yellow (pH 2.5) technique at the light-microscope level and the periodic acid-chromic acid-silver methenamine technique at the electron-microscope level. In paraffin sections, the rabbit and mouse show the appearance of both zona pellucida and follicular fluid in the earliest growing follicles. In the dog, there is a sequence of development with the follicular fluid appearing late, after much of the zona has been secreted. The zona and follicular fluid are highly sulfated in all animals. Zonae pellucidae of atresia appear to lose all traces of sulfation and become highly acidic. At the electronmicroscope level, oocytes contain little if any reactive glycoprotein material which can be related to zona pellucida formation. The initial appearance of the zona material occurs between follicle cell membranes extending outwards and away from the oocyte. Follicle cells of all species consistently contain a variety of reactive Golgi bodies and granules, with exocytotic vesicles, suggesting active synthesis and secretion of zona material. Our observations suggest that in the early stages of oogenesis, the follicular epithelium is responsible for at least part of the synthesis of the zona pellucida. It is possible therefore that both the oocyte and its follicle cells participate, probably on the sequential basis, in the synthesis of the mammalian zona pellucida.     

Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding

To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.     

Immunocytochemical studies on the zona pellucida of cow blastocysts

Labeling of the zona pellucida of cow blastocysts with zona-specific anti-serum shows that antigenicity is unaffected by abnormal cleavage, in vitro culture, or frozen storage. The uniform labeling in thin sections indicates that the zona pellucida is homogeneous antigenically. Heavier labeling of the inner and outer surfaces of the zona pellucida in thick sections appers to be due to greater porosity of these regions, in which the zona material becomes highly dispersed, or even partly solubilized, thereby permitting the formation of an antigen-antibody matrix.     

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.     

Microanatomical diversification of the zona pellucida in aplochelioid killifishes

This study investigates zona pellucida (ZP) ultrastructure in fertilized eggs of annual killifishes (suborder Aplocheiloidei), a group of highly specialized fishes that are able to survive desiccation for several weeks to months before they hatch. Little is known about ZP or chorionic ultrastructure sustaining these life‐history modes, so scanning electron microscopy (SEM) was used to describe this trait in a large number of aplocheiloids with a focus on the family Rivulidae and the genus Hypsolebias. New images of ZP ultrastructure for 52 aplocheiloid species are provided, more than doubling the number characterized thus far. The evolution of chorionic structure within this group is studied using these new data. Characters were coded into a morphological matrix and optimized onto a consensus phylogeny to assess phylogenetic signal and reconstruct ancestral character states. Although ZP characters seem highly homoplastic and exhibit a large amount of structural convergence among lineages, aplocheiloid killifishes have evolved a number of unique structures associated with the chorion. Some annual species seem to have lost long filaments because eggs are deposited in the soil instead of being adhered to aquatic plants.     

Structure of the zona pellucida and cumulus oophorus in three species of native Australian rodents

The sperm head of many Australian hydromyine rodents has three curved hooks projecting from its anterior margin; the structure of the hooks has been characterized, but their function is unknown. In this study, we have investigated whether the hooks might have evolved to assist sperm penetration through more formidable egg vestments, particularly the zona pellucida. Cumulus-oocyte complexes were obtained from two species that possess a three-hooked sperm head (Pseudomys australis and P. nanus) and one species that does not (Notomys alexis) and examined by light and electron microscopy. After fixation in the presence of ruthenium red, the zona pellucida was found to consist of a fibrillar meshwork, but there were no interspecific structural differences. A corona radiata was absent, and the cumulus extracellular matrix was composed of filaments and electron-dense granules in each species. Measurements of the zona thickness in freshly ovulated, unfixed oocytes revealed that it was thinnest (7.8 μm) in P. australis. Which has a three-hooked sperm head, and thickest (11.4 μm) in N. alexis, the species in which the ventral hooks are absent. Hence, no correlation was found between the thickness of the zona pellucida or the structure of the cumulus-oocyte complex, and the presence of three hooks on the sperm head. We conclude, therefore, that it is unlikely that the evolution of the three-hooked sperm head is an adaptation for penetration of increased barriers around the oocyte.     

Localization of epitopes for monoclonal antibodies at the N-terminus of the porcine zona pellucida glycoprotein pZPC

Zona pellucida glycoproteins play an important role in fertilization. In this study, attempts have been made to identify and define epitopes of monoclonal antibodies (mAbs) possessing contraceptive efficacy in vitro. The porcine zona glycoprotein pZPC, a homologue of mouse/human ZP3, was reduced and alkylated and subsequently digested with trypsin. Reverse-phase HPLC of the tryptic digest yielded twenty two peaks (T1–T22). When tested against mAbs reactive against sequential determinants on pZPC, T11 was immunoreactive with two mAbs, mAb-455 and mAb-467, as shown by antigen inhibition ELISA. IC₅₀ values of 3.1 nM and 8.6 nM were recorded versus mAb-455 and mAb-467 respectively, and approximated the IC₅₀ values obtained with intact pZPC. Amino acid analysis, Edman degradation, and FAB-MS identified T11 as the N-blocked decapeptide pyro-Gln-Pro-Val-Trp-Gln-Asp-Glu-Gly-Gln-Arg derived from the N-terminus of pZPC. Synthesis of overlapping octapeptides further identified VWQDE and WQDE as the minimum motifs with antigenie activity for mAb-455 and mAb-467, respectively. Glycine replacement peptides confirmed residues W,Q,E as critical for binding mAb-455 and W,Q,D,E as critical for binding mAb-467. Both mAbs inhibited binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of porcine zona glycoprotein, will assist in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZPC or its homologues in other species. © 1995 wiley-Liss, Inc.     

Light microscopic observations of alterations in staining of the zona pellucida of porcine follicular oocytes: Possible early indication of atresia

Serial sections of porcine ovaries were examined in an attempt to detect early signs of oocyte degeneration/atresia using special staining. Porcine ovaries were fixed in Bouin's fixative and embedded in paraffin using routine techniques. Serial sections (8 μm) were mounted on glass slides and stained with Shorr's S3 and hematoxylin stain. Several criteria were used for examining general histology of the antral follicles: condition of the granulosa layer, antral cavity, the oocyte and its surrounding zona pellucida, and the cumulus layers. A change in the staining characteristic of the zona pellucida was the single most striking observation in all ovaries examined. In presumably healthy follicles, the zona pellucida was uniformly stained green, the granulosa layer was intact with fewer than three pyknotic cells per section, and a uniform basement membrane (stained green) separated the intact theca layers from the remainder of the follicle. In those follicles showing some degree of degenerative changes in the follicular wall, the zona pellucida was stained a bright orange. In the last stages of degeneration, follicles exhibited many pyknotic nuclei throughout the granulosa layers, the layer of granulosa cells was in many cases separated from the basement membrane, and the antrum was infiltrated with lymphocytes. In these follicles, the zona pellucida was always stained orange. Frequently, the zona pellucida was partially stained orange before any detectable changes could be seen in other elements of the follicular wall. Additionally, many non-antral (primary) follicles exhibited oocytes with orange-stained zonae pellucidae. In terminal stages of follicular degeneration, collapsed follicles were infiltrated by connective tissue elements stained bright orange and green. These structures very often contained dying oocytes always with bright orange-stained zonae pellucidae. Scattered throughout the ovarian stroma were many orange-stained remnants of zonae pellucidae. It is thought that perhaps the characteristic staining of the zona pellucida with Shorr's S3 stain may give an early, previously undetectable indication of follicular atresia.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

Participation of the nonreducing terminal beta-galactosyl residues of the neutral N-linked carbohydrate chains of porcine zona pellucida glycoproteins in sperm-egg binding

The zona pellucida (ZP) surrounding the mammalian oocyte is composed of three glycoprotein components (ZPA, ZPB, and ZPC). Mammalian sperm bind to carbohydrate chains of a ZP glycoprotein in the initial phase of fertilization. Sperm-ligand carbohydrate chains have been characterized in mouse, cow, and pig. In pigs, triantennary/tetraantennary neutral complex-type chains from ZPB/ZPC mixture possess stronger sperm-binding activity than those of biantennary chains (Kudo et al., 1998: Eur J Biochem 252:492-499). Most of these oligosaccharides have beta-galactosyl residues at the nonreducing ends. This study used two in vitro competition assays to investigate the participation of the nonreducing terminal beta-galactosyl residues of the ligand active chains in porcine sperm binding. The removal of the nonreducing terminal beta-galactosyl residues from either the ligand active carbohydrate chains or endo-beta-galactosidase-digested glycoproteins significantly reduced their inhibition of sperm-egg binding, indicating that the beta-galactosyl residues at the nonreducing ends are involved in porcine sperm-egg binding. A correlation between the sperm-binding activity and in vitro fertilization rate is also presented.     

Modifications of the lectin binding pattern in the rat zona pellucida after in vivo fertilization

The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. It is involved in the sperm-egg adhesion phenomenon, induces the acrosome reaction, and participates in the late blockage to polyspermy. Thus, during the process of fertilization the cortical reaction is induced and the biochemical and biological properties of the ZP are modified. Some of these changes have been suggested to prevent the polyspermy. However, the mechanisms behind most of these changes are not well understood. Carbohydrate residues of the ZP glycoproteins have been shown to play a key role in the early step of fertilization. In the present study, the changes produced in the terminal oligosaccharide sequences of the rat ZP glycoproteins after in vivo fertilization were investigated by means of lectin-gold cytochemistry. A comparative quantitative analysis of the density of labeling in the ZP before and after fertilization was carried out by automatic counting of gold particles. The ZP of fertilized and unfertilized eggs were labeled by a battery of lectins including PNA, LFA, MAA, AAA, DSA, RCA I, and WGA. For all lectin studied in both fertilized and unfertilized eggs the labeling was preferentially located in the inner region of the ZP. After fertilization, binding of PNA, LFA, MAA, AAA, and DSA decreased in both inner and outer regions of the ZP. Labeling of RCA l-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased in the inner area of the ZP. Digestion of the thin-sections with neuraminidase prior to labeling with WGA resulted in a decrease of labeling for WGA binding sites. However, the labeling density of WGA binding sites was similar in both unfertilized and fertilized eggs upon treatment with neuraminidase. The present results demonstrate that the oligosaccharide chains contained in the rat ZP are modified after fertilization of the oocyte. Cortical granules of the oocytes might be involved in these modifications by two mechanisms: 1) by hydrolysis of terminal carbohydrate residues of ZP glycoproteins by specific glycosidases contained in the granules; and 2) by addition of new glycoproteins to the ZP after the exocytosis of the cortical granules (cortical reaction). © 1996 Wiley-Liss, Inc.     

“Spontaneous” hardening of the zona pellucida of mouse oocytes during in vitro culture

Culture in vitro causes a slow, progressive hardening of the zona pellucida (ZP) of fully grown dictyate oocytes isolated from the mouse ovary. Hardening cannot be prevented by inhibitors of peroxidase or by a tyrosine analogue. Culture in anaerobic conditions is very effective in preventing ZP hardening. If the oocyte is cultured surrounded by its own follicle cells or in contact with cumuli oophori obtained from superovulated females, hardening is much reduced. The results suggest that the “spontaneous” hardening in cultured ovarian oocytes is not due to a cortical reaction, and that a diffusible factor is produced by follicle cells that protect the ZP from hardening.     

Characterization of the functional domains of boar acrosin involved in nonenzymatic binding to homologous zona pellucida glycoproteins

During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida. To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs). The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody. The recombinant truncated β-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation. Three of the five recombinant forms (truncated β-acrosin, Ser/Ala²²²-truncated β-acrosin, and truncated β-acrosin “heavy chain”) had the ability to bind ZPGPs. The two shorter forms (the amino and carboxy termini of truncated β-acrosin) failed to bind. The catalytic site mutant (Ser/Ala²²²) of truncated β-acrosin does not differ from the recombinant truncated β-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process. Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site. Mol. Reprod. Dev. 49:426–434, 1998. © 1998 Wiley-Liss, Inc.